Multiple Sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that affects 250,000-350,000 people in the United States. Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated disorder with clinical and pathological features similar to MS, making it a highly relevant animal model for the study of MS. In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed co-stimulation, is required for complete T cell activation. The B7 family of cell surface molecules is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. It is also known that activated or memory T cells are less dependent on co-stimulation through the B7:CD28/CTLA-4 pathway. Experimental evidence suggests that T cell reactive with myelin components are involved in the pathogenesis of EAE and possibly MS. Building upon our prior work in the EAE model, we will test the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished form naive, naive, myelin-reactive T cells by a lack of dependence upon co-stimulation for activation. To accomplish this, we will examine the requirements for the activation of myelin-reactive T cells over time in both the EAE model and in patients with MS. Using sophisticated techniques in molecular biology such as single strand conformation polymorphism (SSCP) analysis, we will determine whether the co- stimulation-independent, myelin-reactive T cells we observe in vitro are actually expanded in MS patients in vivo, implicating these specific T cells in the pathophysiology of the disease. Our studies will also determine the role of the B7: CD28/CTLA-4 pathway in the activation of disease using T cells in vivo, and their role in the regulation of disease in mice normally resistant to EAE. We will also continue our studies examining mechanisms of peripheral tolerance in the EAE model, focusing on co-stimulatory pathways. The studies proposed will advance our understanding of the pathophysiology of MS and may be applicable for the design of new treatment strategies.